Biology subjects

Enderlein, J.

Publications and source records attributed to Enderlein, J..

31 records · Page 2Linked to original sources

bioRxiv2022

Metal-Induced Energy Transfer (MIET) for Live-Cell Imaging with Fluorescent Proteins

Metal-Induced Energy Transfer (MIET) imaging is an easy-to-implement super-resolution modality that achieves nanometer resolution along the optical axis of a microscope. Although its capability in numerous biological and biophysical studies has been demonstrated, its implementation for live-cell imaging with fluorescent proteins is still lacking. Here, we present its applicability and capabilities for live-cell imaging with fluorescent proteins in diverse cell types (adult human stem cells, human osteo-sarcoma cells, and Dictyostelium discoideum cells), and with various fluorescent proteins (GFP, mScarlet, RFP, YPet). We show that MIET imaging achieves nanometer axial mapping of living cellular and sub-cellular components across multiple timescales, from a few milliseconds to hours, with negligible phototoxic effects.

biophysics↗

bioRxiv2022

Photophysical studies at cryogenic temperature reveal a novel photoswitching mechanism of rsEGFP2

Single-molecule-localization-microscopy (SMLM) at cryogenic temperature opens new avenues to investigate intact biological samples at the nanoscale and perform cryo-correlative studies. Genetically encoded fluorescent proteins (FPs) are markers of choice for cryo-SMLM, but their reduced conformational flexibility below the glass transition temperature hampers efficient photoswitching at low temperature. We investigated cryo-switching of rsEGFP2, one of the most efficient reversibly switchable fluorescent protein at ambient temperature due to facile cis-trans isomerization of the chromophore. UV-visible microspectrophotometry and X-ray crystallography revealed a completely different switching mechanism at [~]110 K. At this cryogenic temperature, on-off photoswitching involves the formation of 2 dark states with blue shifted absorption relative to that of the trans protonated chromophore populated at ambient temperature. Only one of these dark states can be switched back to the fluorescent state by 405 nm light, while both of them are sensitive to UV light at 355 nm. The rsEGFP2 photoswitching mechanism discovered in this work adds to the panoply of known switching mechanisms in fluorescent proteins. It suggests that employing 355 nm light in cryo-SMLM experiments using rsEGFP2 or possibly other FPs could improve the achievable effective labeling efficiency in this technique. Table of Contents artwork O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/504779v1_ufig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@1d5ed02org.highwire.dtl.DTLVardef@1bb2359org.highwire.dtl.DTLVardef@10dc6b1org.highwire.dtl.DTLVardef@16fcd94_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

A DNA-based optical force sensor for live-cell applications

Mechanical forces are relevant for many biological processes, from wound healing or tumour formation to cell migration and differentiation. Cytoskeletal actin is largely responsible for responding to forces and transmitting them in cells, while also maintaining cell shape and integrity. Here, we describe a novel approach to employ a FRET-based DNA force sensor in vitro and in cellulo for non-invasive optical monitoring of intracellular mechanical forces. We use fluorescence lifetime imaging to determine the FRET efficiency of the sensor, which makes the measurement robust against intensity variations. We demonstrate the applicability of the sensor by monitoring cross-linking activity in in vitro actin networks by bulk rheology and confocal microscopy. We further demonstrate that the sensor readily attaches to stress fibers in living cells which opens up the possibility of live-cell force measurements.

biophysics↗

bioRxiv2021

Isotropic Three-Dimensional Dual-Color Super-Resolution Microscopy with Metal-Induced Energy Transfer

Over the last two decades, super-resolution microscopy has seen a tremendous development in speed and resolution, but for most of its methods, there exists a remarkable gap between lateral and axial resolution. Similar to conventional optical microscopy, the axial resolution is by a factor three to five worse than the lateral resolution. One recently developed method to close this gap is metal-induced energy transfer (MIET) imaging which achieves an axial resolution down to nanometers. It exploits the distance dependent quenching of fluorescence when a fluorescent molecule is brought close to a metal surface. In the present manuscript, we combine the extreme axial resolution of MIET imaging with the extraordinary lateral resolution of single-molecule localization microscopy, in particular with direct stochastic optical reconstruction microscopy (dSTORM). This combination allows us to achieve isotropic three-dimensional super-resolution imaging of sub-cellular structures. Moreover, we employed spectral demixing for implementing dualcolor MIET-dSTORM that allows us to image and co-localize, in three dimensions, two different cellular structures simultaneously.

biophysics↗

bioRxiv2021

Electrically Controlling and Optically Observing the Membrane Potential of Supported Lipid Bilayers

Supported lipid bilayers are a well-developed model system for the study of membranes and their associated proteins, such as membrane channels, enzymes, and receptors. These versatile model membranes can be made from various components, ranging from simple synthetic phospholipids to complex mixtures of constituents, mimicking the cell membrane with its relevant physiochemical and molecular phenomena. In addition, the high stability of supported lipid bilayers allows for their study via a wide array of experimental probes. In this work, we describe a platform for supported lipid bilayers that is accessible both electrically and optically. We show that the polarization of the supported membrane can be electrically controlled and optically probed using voltage-sensitive dyes. Membrane polarization dynamics is understood through electrochemical impedance spectroscopy and the analysis of the equivalent electrical circuit. We also describe the effect of the conducting electrode layer on the fluorescence of the optical probe through metal-induced energy transfer. We conclude with a discussion on possible applications of this platform for the study of voltage-dependent membrane proteins and other processes in membrane biology and surface science.

biophysics↗

bioRxiv2021

Mapping Activity-Dependent Quasi-Stationary States of Mitochondrial Membranes with Graphene-Induced Energy Transfer Imaging

Graphene-induced energy transfer (GIET) was recently introduced for the precise localization of fluorescent molecules along the optical axis of a microscope. GIET is based on near-field energy transfer from an optically excited fluorophore to a single sheet of graphene. As a proof-of-concept, we demonstrated its potential by determining the distance between the two leaflets of supported lipid bilayers (SLBs) with sub-nanometer accuracy. Here, we use GIET imaging for three-dimensional reconstruction of the mitochondrial membrane architecture. We map two quasi-stationary states of the inner and outer mitochondrial membranes before and during adenosine tri-phosphate (ATP) synthesis. We trigger the ATP synthesis state in vitro by activating mitochondria with precursor molecules. Our results demonstrate that the inner membrane (IM) approaches the outer membrane (OM) while the outer membrane (OM) does not show a measurable change in average axial position upon activation. As a result, the inter-membrane space (IM-OM distance) is reduced by [~]2 nm upon activation of the mitochondria. This direct experimental observation of the subtle dynamics of mitochondrial membranes and the change in inter-membrane distance induced by ATP synthesis is relevant for our understanding of the physical functioning of mitochondria.

biophysics↗

bioRxiv2021

Instant three color multi-plane fluorescence microscopy

One of the most widely used microscopy techniques in biology and medicine is fluorescence microscopy, offering high specificity in labeling as well as maximum sensitivity. For live cell imaging, the ideal fluorescence microscope should offer high spatial resolution, fast image acquisition, three-dimensional sectioning, and multi-color detection. However, most existing fluorescence microscopes have to compromise between these different requirements. Here, we present a multi-plane multi-color wide-field microscope that uses a dedicated beam-splitter for recording volumetric data in eight focal planes and for three emission colors with frame rates of hundreds of volumes per second. We demonstrate the efficiency and performance of our system by three-dimensional imaging of multiply labeled fixed and living cells.

biophysics↗

bioRxiv2020

Confocal Laser-Scanning Fluorescence-Lifetime Single-Molecule Localisation Microscopy

Fluorescence lifetime imaging microscopy (FLIM) is an important technique that adds another dimension to the intensity and colour information of conventional microscopy. In particular, it allows for multiplexing fluorescent labels that have otherwise similar spectral properties. Currently, the only super-resolution technique that is capable of recording super-resolved images with lifetime information is STimulated Emission Depletion (STED) microscopy. In contrast, all Single-Molecule Localisation Microscopy (SMLM) techniques that employ wide-field cameras completely lack the lifetime dimension. Here, we combine Fluorescence-Lifetime Confocal Laser-Scanning Microscopy (FL-CLSM) with SMLM for realising single-molecule localisation-based fluorescence-lifetime super-resolution imaging (FL-SMLM). Besides yielding images with a spatial resolution much beyond the diffraction limit, it determines the fluorescence lifetime of all localised molecules. We validate our technique by applying it to direct STochastic Optical Reconstruction Microscopy (dSTORM) and Points Accumulation for Imaging in Nanoscale Topography (PAINT) imaging of fixed cells, and we demonstrate its multiplexing capability on samples with two different labels that differ only by fluorescence lifetime but not by their spectral properties.

biophysics↗

bioRxiv2020

Three-dimensional beating dynamics of Chlamydomonas flagella

Axonemes are the basic structure of motile cilia and flagella, and the investigation of how they function and move requires rapid three-dimensional imaging. We built a multi-plane phase-contrast microscope for imaging the three-dimensional motion of unlabeled flagella of the model organism Chlamydomonas reinhardtii with sub-m spatial and 4 ms temporal resolution. This allows us to observe not only bending but also the three-dimensional torsional dynamics of these small structures. We observe that flagella swim counter-clockwise close to a surface, with negatively-valued torsion at their basal and positively-valued torsion at their distal tips. To explain the torsional dynamics and signature, we suggest the existence of an intrinsic negative twist at the basal end that is untwisted by active positive-twist-inducing dynein motor proteins. Moreover, dyneins walking towards the basal induce an opposite twist at the distal tip. Bending of the whole axoneme structure then translates this twist into an observable torsion. This interconnection between chiral structure, twist, curvature, and torsion is fundamental for understanding flagellar mechanics.

biophysics↗

bioRxiv2020

Gradual Compaction of the Nascent Peptide During Cotranslational Folding on the Ribosome

ABSTRACTNascent polypeptides begin to fold in the constrained space of the ribosomal peptide exit tunnel. Here we use force profile analysis (FPA) and photo-induced energy-transfer fluorescence correlation spectroscopy (PET-FCS) to show how a small α-helical domain, the N-terminal domain of HemK, folds cotranslationally. Compaction starts vectorially as soon as the first α-helical segments are synthesized. As nascent chain grows, emerging helical segments dock onto each other and continue to rearrange at the vicinity of the ribosome. Inside or in the proximity of the ribosome, the nascent peptide undergoes structural fluctuations on the μs time scale. The fluctuations slow down as the domain moves away from the ribosome. Folding mutations have little effect on folding within the exit tunnel, but abolish the final domain stabilization. The results show the power of FPA and PET-FCS in solving the trajectory of cotranslational protein folding and in characterizing the dynamic properties of folding intermediates.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2019

Dimerization of human drebrin-like protein governs its biological activity

Drebrin-like protein (DBNL) is a multidomain F-actin binding protein, which also interacts with other molecules within different intracellular pathways. Here, we present quantitative measurements on size and conformation of human DBNL. Using dual focus fluorescence correlation spectroscopy, we determined the hydrodynamic radius of DBNL monomer. Native gel electrophoresis and dual color fluorescence cross-correlation spectroscopy show that both endogenous and recombinant DBNL exist as dimer under physiological conditions. We demonstrate that C-terminal truncations of DBNL downstream of the coiled-coil domain result in its oligomerization at nanomolar concentration. In contrast, the ADF-H domain alone is a monomer, which displays a concentration-dependent self-assembly. In vivo FLIM-FRET imaging shows that the presence of only actin-binding domains is not sufficient for DBNL to localize properly at actin filament inside the cell. In summary, our work provides a detailed insight on structure-function relationship of human drebrin-like protein.

biophysics↗

bioRxiv2018

Nanobody Detection of Standard Fluorescent Proteins Enables Multi-Target DNA-PAINT with High Resolution and Minimal Displacement Errors

DNA-PAINT is a rapidly developing fluorescence super-resolution technique which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20-25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using 3 nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the 3 targets with 20 nm resolution, and within only 35 minutes acquisition time.

cell biology↗

bioRxiv2018

Photon yield enhancement of red fluorophores at cryogenic temperatures

Single Molecule Localization Microscopy has become one of the most successful and widely applied methods of Super-resolution Fluorescence Microscopy. Its achievable resolution strongly depends on the number of detectable photons from a single molecule until photobleaching. By cooling a sample from room temperature down to liquid nitrogen temperatures, the photostability of dyes can be enhanced by more than 100 fold, which results in an improvement in localization precision greater than 10 times. Here, we investigate a variety of fluorescent dyes in the red spectral region, and we find an average photon yield between 3.5 {middle dot} 106 to 11 {middle dot} 106 photons before bleaching at liquid nitrogen temperatures, corresponding to a theoretical localization precision around 0.1 nm.

biophysics↗